Eriocaulaceae

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Botanical Journal of the Linnean Society, 2013, 171, 225–243. With 5 ﬁgures

Molecular phylogenetics and biogeography of Neotropical Paepalanthoideae with emphasis on Brazilian Paepalanthus (Eriocaulaceae) Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/1/225/2557485 by guest on 06 January 2020 MARCELO TROVÓ1,2*, MARIA JOSÉ GOMES DE ANDRADE3, PAULO TAKEO SANO1, PATRÍCIA LUZ RIBEIRO3 and CÁSSIO VAN DEN BERG3

1Laboratório de Sistemática Vegetal, Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, Rua do Matão 277, Cidade Universitária, CEP 05508-900, São Paulo, Brazil 2Evolution and Biodiversity of Plants, Faculty for Biology and Biotechnology, Ruhr University Bochum, D-44780 Bochum, Germany 3Laboratório de Sistemática Molecular de Plantas, Departamento de Ciências Biológicas, Universidade Estadual de Feira de Santana, Av. Transnordestina s.n., CEP 44036-900, Feira de Santana, Brazil

Received 10 January 2012; revised 25 June 2012; accepted for publication 29 August 2012

Subfamily Paepalanthoideae encompass the largest generic diversity in Eriocaulaceae. In the present study, the main goals were to infer the phylogeny of this subfamily focusing on Paepalanthus, to evaluate recent classifications and morphological characters in a phylogenetic context and to reconstruct the historical biogeography of the group. Sampling involved 94 ingroup species corresponding to all recognized genera and three outgroup species. Two molecular data sets, nuclear ribosomal internal transcribed spacer (nrITS) and plastid trnL-trnF, were analysed under parsimony and Bayesian methods. Rondonanthus is monophyletic and confirmed as sister to the remaining Paepalanthoideae. Leiothrix and Actinocephalus are each monophyletic, whereas Syngonanthus may be either monophyletic or paraphyletic with the recognition of Philodice. Four subgenera of Paepalanthus are monophyletic, but P. subgenus Paepalanthus is polyphyletic. Morphological characters used in previous classifications are assessed as putative synapomorphies for recognized genera. Some of the characters employed in defining Paepalanthus subcategories appear to have evolved multiple times, and many clades may be exclusively defined by molecular synapomorphies. Biogeographical reconstructions suggest that the current distribution patterns may be related to vicariance and a few long-distance dispersal events. Furthermore, some clades are restricted to narrow geographical areas, perhaps important as a means of conserving evolutionary processes. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 225–243.

ADDITIONAL KEYWORDS: internal transcribed spacer (ITS) – morphology – South America – systematics – taxonomy – trnL-trnF.

INTRODUCTION Hensold, 1990; Stützel, 1998; Sano, 2004; Parra et al., 2010; Andrade et al., 2011). The family is historically Eriocaulaceae comprise c. 1200 species and 11 genera divided into two subfamilies: Paepalanthoideae and distributed throughout the tropics (Giulietti & Eriocauloideae (Koernicke, 1863; Ruhland, 1903). The former is characterized as possessing haplostemonous *Corresponding author. Current address: Departamento de androecia, eglandular corollas and gynoecia with Botânica, Instituto de Biologia, Universidade Federal do Rio alternating stigmatic and nectariferous branches de Janeiro, CCS, Bloco A1, Cidade Universitária, Ilha do Fundão, CEP 21941-590, Rio de Janeiro, RJ, Brasil. E-mail: (Ruhland, 1903; Rosa & Scatena, 2007). All nine [email protected] genera and c. 800 species of Paepalanthoideae

(Fig. 1) are predominantly distributed throughout the Andrade, 2007; Andrade et al., 2010) was fairly Neotropics (Giulietti & Hensold, 1990; Stützel, 1998). similar: all confirming the monophyly of both sub- According to Giulietti et al. (2005), c. 95% of the families and the genera Leiothrix Ruhland and species included in this subfamily are narrow endem- Actinocephalus (Koern.) Sano emend. (F. N. Costa & ics and restricted to a few small populations. Some P. T. Sano, unpubl. data; including Paepalanthus sub- species are of great economic importance. Every section Aphorocaulon Ruhland) and the polyphyly of year, large numbers of inflorescences from species of Paepalanthus. Thus, based on the monophyly of Syngonanthus Ruhland, Comanthera L.B.Sm. and Paepalanthoideae (Giulietti et al., 1995; Unwin, 2004; Paepalanthus Mart. are exported to North America Andrade, 2007; Andrade et al., 2010), our main goal and Europe as ‘everlasting plants’ and, more recently, was to infer phylogenetic relationships in the sub- Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/1/225/2557485 by guest on 06 January 2020 as ‘golden grass’ (Giulietti et al., 1988; Schmidt, family, with an emphasis on Paepalanthus and its Figueiredo & Scariot, 2007; Costa, Trovó & Sano, Brazilian subgroups. Evolution of morphological char- 2008; Parra et al., 2010). These species are not culti- acters and biogeographic history of the Paepalanthoi- vated but simply harvested in situ, leading to deple- deae were reconstructed in order to clarify the tion of the natural populations. Indeed, many species classification and evolution of the group in the of Paepalanthoideae are highly endangered. For Neotropics. example, 94 species have been cited on the Red List of State of Minas Gerais and 20 on the Red List of Brazil (Biodiversitas, 2008). Paepalanthus spp. are also MATERIAL AND METHODS notable for their antioxidant and mutagenic proper- TAXA SAMPLED ties (Varanda et al., 2006; Devienne et al., 2007). In Paepalanthoideae, Paepalanthus is the largest All nine genera of Paepalanthoideae and all infrage- genus with c. 380 species (Giulietti & Hensold, 1990; neric categories of Paepalanthus recognized by Stützel, 1998). Morphological and species diversity Ruhland (1903) were sampled, except for Paepalan- is concentrated in Brazilian campos rupestres in the thus subgenus Psilandra Ruhland, known exclusively Espinhaço Range and in the Guayana Shield High- from the type specimen. Overall, 94 species were lands (Giulietti & Hensold, 1990; Hensold, 1991, sampled as follows: Actinocephalus 5/28 (species 1999). The genus is characterized by pistillate sampled/species belonging to the genus), Comanthera flowers with free petals and stigmatic/nectariferous 3/37, Lachnocaulon Kunth 1/7, Leiothrix 6/64, Paepal- branches that are free at the same insertion point. anthus 73/~380, Philodice Mart. 1/2, Rondonanthus Nevertheless, with the exception of these few floral Herzog 2/6, Syngonanthus 2/~150 and Tonina Aubl. characters, morphological variation in both floral 1/1. The sampling in Paepalanthus and Syngonanthus and vegetative traits is broad. This variation pro- is limited at a first glance. However, many species of vided the basis for the proposition of > 20 infrage- these genera are known only from the type specimen neric categories in previous classifications of the or unknown type localities. Recent taxonomic revi- genus (Koernicke, 1863; Ruhland, 1903). Some cat- sions identified many synonyms in these groups, and egories, such as Paepalanthus subgenus Platycaulon the number of species of Paepalanthus and Syngo- Koern., are morphologically easily recognized, nanthus may actually be much smaller. Moreover, whereas others, such as Paepalanthus series Paepal- Syngonanthus is monomorphic in relation to the anthus, simply represent aggregations of possibly morphological traits evaluated (Koernicke, 1863; unrelated species (Stützel, 1998). Thus, the exten- Ruhland, 1903). Three species of Eriocaulon L. were sive morphological variation in Paepalanthus may included as outgroups, based on previous phylogenies obscure the phylogenetic relationships and explain, (Unwin, 2004; Andrade et al., 2010). The complete list at least in part, its complex nomenclatural and of taxa and voucher specimens is given in Appendix 1. taxonomic history. The first phylogenetic study involving Paepalan- thoideae was based entirely on morphological data SEQUENCES AND ALIGNMENT (Giulietti, Amaral & Bittrich, 1995). In this study, The nuclear internal transcribed spacer (ITS) region Paepalanthoideae emerged as monophyletic, whereas and the plastid trnL-trnF region were chosen because Paepalanthus was polyphyletic. In a later study, of their rate of sequence variation, based on previous Giulietti et al. (2000), taking into consideration mor- studies in the family by Unwin (2004), Andrade phology, anatomy and chemistry, concluded that (2007) and Andrade et al. (2010). Sequences for 59 Paepalanthoideae are paraphyletic, but many of the species were obtained from Andrade et al. (2010) and genera are monophyletic. Polyphyly of Paepalanthus additional sequences for 35 species were obtained for was also stated in that study. The outcome of recent this study. All GenBank accession numbers are listed molecular phylogenetic analyses (Unwin, 2004; in Appendix 1.

© 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 225–243 EVOLUTION OF PAEPALANTHOIDEAE 227 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/1/225/2557485 by guest on 06 January 2020

Figure 1. Photographs of Paepalanthoideae representatives in nature. A, Actinocephalus polyanthus (Bong.) Sano. B, Comanthera linearis (Ruhland) L.R. Parra & Giul. C, Leiothrix crassifolia (Bong.) Ruhland. D, Paepalanthus chiquitensis Herzog. E, Rondonanthus roraimae (Oliver) Herzog. F, Syngonanthus nitens (Bong.) Ruhland. G, Tonina ﬂuviatilis Aubl. Scale bars, 5 cm (A); 4 cm (B); 0.5 cm (C); 25 cm (D); 7 cm (E); 0.4 cm (F); 0.3 cm (G). [Photographs: M. Trovó (A, C, D and F); L. Echternacht (B and E); S. Martins (G)].

Total DNA was extracted from leaves dried in the same parameters applied for the initial tree silica gel, using 5–50 mg and a modified 2 ¥ cetyl search (Felsenstein, 1985). Gaps were treated as trimethylammonium bromide (CTAB) protocol (Doyle missing data. Multiple equally most-parsimonious & Doyle, 1987). The trnL-trnF spacer was amplified trees were summarized as strict consensus trees and sequenced using the c and f universal primers (Sokal & Rohlf, 1981). (Taberlet et al., 1991). Amplification was conducted For Bayesian analyses, evolutionary models for on 50 mL reaction containing 1 ¥ polymerase chain the molecular data were chosen with hLRTs using reaction (PCR) reaction buffer, 2.5 mM magnesium MrModeltest 2.2 (Nylander, 2004). Model selection chloride (MgCl2), 0.2 mM deoxyribonucleotide triphos- was conducted for the ITS region split into three phates (dNTPs), 0.5 mM each primer, 0.5 mg bovine parts, corresponding to ITS1, 5.8S and the ITS2 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/1/225/2557485 by guest on 06 January 2020 serum albumin (BSA) and 0.25 units Taq DNA regions, and the trnL-trnF region split in two, corre- polymerase (Phoneutria Ltd, Belo Horizonte, Brazil). sponding to the intron and spacer regions. Analyses PCR included an initial denaturing cycle at 94 °C for were performed on MrBayes 3.1 (Ronquist & 3 min, followed by 35 cycles at 94 °C for 1 min, Huelsenbeck, 2003), run for 5 000 000 generations 50–52 °C for 1 min and 72 °C for 2 min, and a final using two sets of four chains, sampling one tree every extension of 72 °C for 5 min. The ITS region was 500 generations. Instead of selecting the burn-in amplified and sequenced using primers 75 and 92 for using the graphic approach by plotting the likelihood angiosperms (Desfeaux et al., 1996). ITS amplification scores against the number of generations, we dis- was with a 50-mL reaction containing 1 ¥ PCR reac- carded the trees obtained before the standard devia- tion buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, 0.5 mM each tions between two sets of chains reached a value primer, 1 mg BSA, 1.0 M betaine, 2% dimethyl sul- < 0.01. We consider this a stricter way of guarantee- phoxide and 0.25 units Taq DNA polymerase. PCR ing that chains achieved stability. A majority rule included an initial denaturing cycle at 94 °C for consensus of the remaining trees was obtained in 3 min, followed by 35 cycles of 94 °C for 45 s, 56–58 °C PAUP*, with group frequencies representing the for 1 min and 72 °C for 2 min, and a final extension of posterior probabilities. 72 °C for 5 min. PCR products were purified by enzymatic treat- ment using the EXOSAP-IT kit (GE Healthcare, USB, CHARACTER AND BIOGEOGRAPHICAL Cleveland, OH, USA). Sequencing reactions were RECONSTRUCTION carried out with Big Dye Terminator kit, ver. 3.1 All specimens were analysed under a stereomicro- (Applied Biosystems, Foster City, CA, USA). Samples scope in order to evaluate the morphological charac- were sequenced in both directions using the same ters, especially those that are taxonomically relevant. primers in an ABI 3130XL automatic sequencer, Thirteen characters were coded into binary states and according to protocols supplied by the manufacturer. mapped onto the consensus tree of the combined Sequences were edited using the STADEN package molecular data set using ‘Trace Character History’ (Staden, Beal & Bonfiel, 1998) and aligned using implemented in Mesquite ver. 2.74 for Macintosh MUSCLE (Edgar, 2004), with subsequent visual cor- (Maddison & Maddison, 2009). For this, all characters rection. The molecular data sets ITS and trnL-F were were treated as unordered and optimization with analysed independently and simultaneously. The accelerated transformations (ACCTRAN) was used. combined molecular matrix included only terminals Tree and character distributions were edited and sequenced for both markers. All molecular matrices summarized using Figtree 1.3.1 for Macintosh are available at TreeBase http://www.treebase.org (Rambaut, 2009), facilitating the visualization of all (study no. 10836). character state distributions. All characters and their respective states and the morphological matrix encompassing all species in the combined analysis are PHYLOGENETIC ANALYSES available in Appendix 2. Parsimony analysis was conducted in PAUP* 4.0b10 Distributions of all species were established accord- (Swofford, 2002). One thousand replicates with ing to recent taxonomic revisions and herbarium random taxon–addition were conducted with tree– searches (Appendix 3). They were coded as binary bisection–reconnection (TBR). Up to 250 trees were states in a matrix according to the following a priori retained for each replicate. For combined analysis, defined regions (outlined together with the results in the partition homogeneity test implemented in PAUP Fig. 5): Espinhaço Range in Minas Gerais (A); Espin- 4.0b10 (Swofford, 2002) including both partitions was haço Range in Bahia (B); cerrado (C); Lowland run with 1000 homogeneity replicates, saving a Guianan savanna (D); restingas (E); tepuis (F); and maximum of 1000 trees. Measurements of clade Caribbean (G). Biogeographical reconstructions were support were estimated by bootstrap analyses using performed using S-DIVA–RASP ver. 1.107 (Ronquist,

Table 1. Statistics for analyses of trnL-trnF and internal Rondonanthus is monophyletic (89/95, bootstrap/ transcribed spacer (ITS) data sets for Paepalanthoideae Bayesian posterior probabilities) (see also Supporting Information, Appendix S1), has linear staminodes trnL- trnL-trnF, (character: state 6:1) and is sister to clade B (100/ Characteristic trnF ITS ITS 100). The clade including Rondonanthus and the remaining genera is characterized by the absence of No. of taxa 89 90 82 glands on the staminate ﬂower (2:1), the haploste- Aligned length 1372 998 2370 monous androecium (3:1) and absence of leaf fenes- Constant characters 740 397 1142 tration (4:1). The monophyly of clade B (91/97) is

Variable characters 382 535 909 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/1/225/2557485 by guest on 06 January 2020 supported by the tubular corolla in the staminate Informative characters 270 428 690 flower (5:1). Leiothrix (100/100) is sister to clade C No. of trees obtained* 2509 165 112 (90/99), which contains clade D (100/94) and the core Minimum tree length* 807 1798 2599 Consistency index 0.642 0.531 0.565 Paepalanthus clade (100/100) (Fig. 2; see also Sup- porting Information, Appendix S2). Clade D consists *Statistics for parsimony. of Syngonanthus spp. sister to Philodice (100/100), together forming a monophyletic group with the recently re-established Comanthera (100/100). The 1997; Nylander et al., 2008; Yu, Harris & He, 2011) relationship between these three genera is supported using the last 8000 trees from the post burn-in by the median fusion of the pistillate flower (8:1). sample, over the combined Bayesian tree. Polytomies There are two distinct lineages in the core Paepalan- were solved manually but not interpreted in the final thus clade, clades E (95/100) and F (90/95) (Fig. 2; see reconstruction. Frequencies of ancestral area recon- also Supporting Information, Appendix S2). Clade E structions are described for each tree node. is composed of a basal dichotomy with Tonina/ Lachnocaulon (100/100) (Supporting Information, Appendix S1) and species of Paepalanthus and Actinocephalus (90/100). The relationships among RESULTS these species may vary according to the different Statistics for analyses of the trnL-F and ITS data sets analyses (Fig. 2; see also Supporting Information, are given in Table 1. The partition homogeneity test Appendix S1). Both species of Paepalanthus subgenus indicated no significant disagreement between trnL-F Thelxinöe Ruhland form a strongly supported clade and ITS regions (P = 0.10). The evolutionary model (100/100) and emerge in clade G, (85/100). In clade G, selected for ITS1, ITS2, the trnL intron and the Actinocephalus emerges as sister to a clade, compris- trnL-trnF spacer was GTR + I + G; for 5.8S K80 + G ing Paepalanthus section Diphyomene Ruhland and was selected. For trnL-F the following characters Paepalanthus series Dimeri (Ruhland) Giul. were excluded in the aligned matrix: 1–55, 550–590, In two analyses (Fig. 2; see also Supporting Infor- 910–940, 1250–1280 and 1280–1372; for ITS 1–40, mation, Appendix S1), Paepalanthus subgenus Mono- 507–518, 791–802 and 996–998 were excluded. These sperma Hensold (100/100) is sister to the remaining regions were excessively variable and, consequently, species in clade F (90/95) and has only the median we were not confident in the alignments achieved. locule of the gynoecium fertile (7:1). In clade H (100/ Variation in the ITS region was greater than in trnL- 100), clade I (90/97) includes Paepalanthus erigeron trnF (47.3 vs. 25.9%). In addition, a comparison Mart., the type species of Paepalanthus. Clade J among tree topologies from all the analyses revealed (–/95) is split in two main lineages. In clade K (90/ high congruence, with no supported disagreement 100), P. subgenus Platycaulon is monophyletic between the topologies derived from individual or (Fig. 2; see also Supporting Information, Appendix combined data sets. Therefore, the trees derived from S2) (100/100), defined by the fused scapes (11:1), and individual analyses are presented in the Supporting is sister (98/100) to P. subgenus Xeractis Mart. (85/ Information (Appendix S1 and Appendix S2). In 91) (Fig. 2; see also Supporting Information, Appen- either individual or combined trees, the main clades dix S1), which is defined by the hairs on the adaxial are supported by high bootstrap values and high surface of the staminate flower corolla (9:1). Paepal- Bayesian posterior probabilities. The main results of anthus subsection Dichocladus is sister (85/91) to the combined analyses (Fig. 2), the morphological species originally placed in the recently synomized character optimizations (Figs 3, 4) and the biogeo- Blastocaulon Ruhland. Clade L (92/96) comprises graphical reconstruction (Fig. 5) are detailed below. several infrageneric categories of Paepalanthus. In the few cases of discrepancies between tree topolo- Biogeographical reconstruction of Paepalanthoi- gies, Appendix S1 and/or Appendix S2 (see Supporting deae is presented in Figure 5. Although Rondonan- Information) are also cited. thus is restricted to the tepuis and adjacent

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Figure 2. The 50% majority rule consensus tree from the Bayesian analysis of the combined trnL-trnF and internal transcribed spacer (ITS) data sets. Numbers above branches indicate bootstrap support and numbers below indicate Bayesian posterior probabilities. Arrowheads indicate clades not recovered in the parsimony analysis. The shaded box indicates the core Paepalanthus clade, in which white boxes are correlated with biogeographical patterns. Clade names follow the classiﬁcation of Ruhland (1903). Branches marked with grey lines have simple carinal stigmas, branches marked with dark dotted lines have simple commissural stigmas, branches marked with dark plain lines have biﬁd commissural stigmas and branches marked with white dotted lines have an equivocal character state.

2007; Andrade et al., 2010). After re-rooting their tree (Giulietti et al., 2000: 585) between Paepalanthoideae and Eriocauloideae, similarities with our conclusions are evident. The existence of a clade comprising Paepalanthus, Actinocephalus, Tonina and Lachno- caulon can be confirmed, with the exception of the sister relationship between Philodice and Tonina. The existence of a clade including Actinocephalus and some species of Paepalanthus with dimerous flowers can also be confirmed. Nevertheless, the relationship Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/1/225/2557485 by guest on 06 January 2020 between Leiothrix and Syngonanthus needs to be re-evaluated, as it is again not supported here, but only in one of the cladograms of Andrade et al. (2010: 382). Similarities with previous molecular phylogenetic analyses (Unwin, 2004; Andrade, 2007; Andrade et al., 2010) are not surprising, attributable to the Figure 3. Character distribution of six morphological previous levels of support of previous studies and characters onto the summarized combined molecular phy- the use of the same molecular markers and part of the logenetic tree shown in Figure 2. Characters are: 2, glands data set of Andrade (2007) and Andrade et al. (2010). on staminate flower; 3, androecium; 4, leaf fenestration; 5, The main similarities are the monophyly of Leiothrix, petals of the staminate flower; 6, staminodes on pistillate the sister-group relationship between Tonina and flower; 7, fertile locules of the gynoecium. Grey squares Lachnocaulon and the formation of two main lineages represent the state 0, black squares represent the state 1. in the core Paepalanthus clade, one formed by species Numbers above the character bars indicate the character from Paepalanthus, Actinocephalus, Tonina and on Appendix 2 and numbers below the character bars Lachnocaulon and the other by species recognized as indicate the character state transition. Paepalanthus, including the type species. There are, however, some differences between the lowlands Guianan savannas, the ancestor of clade A present study and results from previous analyses is not confidently reconstructed to a specific area. (Andrade, 2007; Andrade et al., 2010). The main dif- The Espinhaço Range in Minas Gerais, which is the ferences are that a monophyletic Rondonanthus was primary centre of diversity, is consistently recovered sister to the remaining Paepalanthoideae and that as ancestor for many clades. In clade E, with uncer- Comanthera and not Leiothrix was sister to a clade tain ancestral distribution, emerge species restricted that includes Syngonanthus and Philodice. These to the Espinhaço Range, mainly in Minas Gerais, differences may arise as a result of alignment dif- and a clade optimized to the savannas. Subclades ferences and increased sampling. They are in accord- include one formed by Actinocephalus, diversified in ance with morphological evidence, as the included the Espinhaço Range, and a clade with dimerous Rondonanthus spp. have free petals on the stami- flowers, diversified in the cerrado and lowland nate flowers at maturity. However, the remaining Guianan savannas. In clade F, the biogeographical species of the genus, which were not sampled, have signal is significant. A clade restricted to the tepuis a typical tubular corolla. The inclusion of such is sister to a clade restricted mainly to the Espin- species will be crucial to further evaluate this char- haço Range (clade H). In the Espinhaço Range clade, acter and the monophyly of the group itself. Syngo- two distinct lineages are clearly recognized, one nanthus, Philodice and Comanthera share pistillate associated with the Espinhaço Range in Bahia flowers with petals fused in the middle. The inclu- (clade I) and one with the Espinhaço Range in sion of more terminals, especially of Paepalanthus, Minas Gerais (clade J). may increase the clade support and the resolution in the core Paepalanthus clade. In this clade, four of the five subgenera of Paepalanthus are mono- DISCUSSION phyletic, but P. subgenus Paepalanthus is polyphy- PHYLOGENETIC IMPLICATIONS letic. Clade F comprises P. subgenus Monosperma, restricted to the tepuis of northern South America, Based on morphological and chemical data, Giulietti and sister to a clade comprising two subclades, one et al. (2000) concluded that Paepalanthoideae were restricted to the north of the Espinhaço Range and paraphyletic, a hypothesis not confirmed here or in the other restricted to the south of the Espinhaço previous molecular analyses (Unwin, 2004; Andrade, Range.

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Figure 4. Character distribution of six morphological characters onto the summarized combined molecular phylogenetic tree shown in Figure 2. Characters are: 8, petals of the pistillate flower; 9, trichomes inside the corolla of the staminate flower; 10, involucral bracts adaxial surface; 11, scapes; 12, involucral bracts; 13, floral merism. Grey squares represent state 0, black squares represent state 1. Numbers above the character bars indicate the character on Appendix 2 and numbers below the character bars indicate the character state transition.

MORPHOLOGICAL IMPLICATIONS phies. Rondonanthus is defined by the presence of The monophyly of Paepalanthoideae suggests several linear staminodes (Fig. 3). The position of this genus putative synapomorphies for the group, including as an early divergent genus in Paepalanthoideae was central projection of the carpels arising on nectarif- previously suggested by Hensold & Giulietti (1991). erous branches, lateral projections of the carpels Some characters, such as the linear staminodes in the arising on stigmatic branches (Fig. 2), eglandular pistillate flower and the bisexual flower in R. flabel- petals and a haplostemonous androecium (Fig. 3). liformis (Moldenke) Hensold & Giulietti could be Most species of this subfamily have unfenestrated interpreted as plesiomorphies. In Eriocaulaceae, the leaves (Fig. 3). Based on current sampling, clade B linear staminodes are best interpreted as a synapo- may be defined as having the petals of the staminate morphy, the first steps in the reduction of one of the flower forming a tubular corolla (Fig. 3). However, two whorls of stamens. In Paepalanthoideae, the this character is shared with Mesanthemum Koern., presence of the linear staminodes may, however, be all species of Rondonanthus except Rondonanthus regarded as plesiomorphic as they are lost in descend- capillaceus (Koern.) Hensold & Giul. and it is absent ant clades. The inclusion of R. flabelliformis (not in some unsampled species, such as Leiothrix fluitans sampled) will be also crucial in evaluating the evolu- (Mart.) Ruhland. tion of this character, because of its supposedly Several characters commonly used to separate the unique bisexual flowers, with apparently functional genera of Eriocaulaceae may be putative synapomor- stamens. That species will be also important for

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Figure 5. Graphical representation of ancestral distributions reconstructed over the combined Bayesian tree for subfamily Paepalanthoideae. The pie charts on each node show the relative probabilities of ancestral distribution obtained from the last 8000 Bayesian post-burn-in trees. States of extant species and colour correspondence with the respective areas can be assessed directly from the terminals. Biogeographical regions: (1) Espinhaço Range in Minas Gerais; (2) Espinhaço Range in Bahia; (3) cerrado; (4) Lowland Guianan savannas; (5) restingas; (6) tepuis; and (7) Caribbean. testing the monophyly of the genus itself and to Leiothrix has nectariferous and stigmatic branches test the validity of the morphological placement of inserted at the style at different levels and the pres- Wurdackia Moldenke in Rondonanthus (Hensold & ence of basiﬁxed anthers (shared with Tonina)as Giulietti, 1991). putative synapomorphies (Andrade et al., 2010).

Fused petals in the mid-region of pistillate flowers, the phylogeny, the focus has to be on key species that also a feature in Mesanthemum (Eriocauloideae) and now form morphologically heterogeneous clades. In three unsampled Rondonanthus spp., are a synapo- contrast, further sampling in well-supported and mor- morphy for clade D, containing Comanthera, Syn- phologically well-defined clades such as P. subgenera gonanthus and Philodice (Fig. 4). In this clade, Platycaulon and. Xeractis and Actinocephalus may Syngonanthus appears monophyletic and is sister to not be necessary to elucidate further the intergeneric Philodice; however, Syngonanthus is poorly sampled relationships in Paepalanthus. so far. The Tonina/Lachnocaulon clade may be defined Other important features, such as relative size of as having smaller petals in pistillate flowers. In involucral bracts and floral merism (characters 12 Tonina, this reduction is only partial, resulting in a and 13) seem to have evolved multiple times in the Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/1/225/2557485 by guest on 06 January 2020 small petal with long trichomes, whereas in Lachno- group (Fig. 4) and appear in many clades. The flowers caulon the reduction to many trichomes is complete are usually trimerous in most Paepalanthoideae, but (Ruhland, 1903; Unwin, 2004; Andrade et al., 2010). are dimerous in a few species (Koernicke, 1863; In Paepalanthus, certain characters used to distin- Ruhland, 1903). Dimerous flowers may have evolved guish infrageneric categories may also constitute at least twice, in Paepalanthus sphaerocephalus clade synapomorphies. Paepalanthus subgen. Xeractis Ruhland and in clade G. As far as is known from can be defined by the hairs on the adaxial surface of developmental studies (Stützel, 1984, 1985) the pre- the corolla in staminate flowers as a putative synapo- condition for this shift to dimery is the late initiation morphy (Fig. 4). Some Eriocaulon spp., such as Erio- and subsequent reduction of the median sepal. The caulon linearifolium Koern. and Eriocaulon parallel shift to dimery in different genera, such as modestum Kunth, both included as outgroups, are Paepalanthus, is therefore not surprising. In clade G, described as possessing similar trichomes. However, only Actinocephalus has trimerous flowers, probably in the specimens analysed, such trichomes are attributable to a reversion. The inclusion of more lacking. The inclusion of a few more species, such as dimerous species and studies on their floral develop- the rare Paepalanthus aureus Silveira, would be rel- ment would be of interest in determining the evolu- evant to test the validity of the hairs on the adaxial tion of this character. The relative size of the floral surface of the bracts (10:1) as a putative synapomor- bracts was considered an important feature in defin- phy (Fig. 4). This species does not display this feature ing groups such as P. subgenus Xeractis (Ruhland, (Hensold, 1988) and, in a recent phylogenetic analy- 1903). They evolved, however, in different genera and sis, Echternacht et al. (2011) indicate that this char- even in Paepalanthus appear in unrelated species, as acter is not uniform in the group and should be predicted by Hensold (1988) and Echternacht et al. studied in greater depth. Paepalanthus subgenus (2011). Platycaulon is defined by the fusion of scapes at Various groups in the core Paepalanthus clade are various levels (Fig. 4) (Tissot-Squalli, 1997), and in P. defined exclusively by molecular characters. Search- subgenus Monosperma, the achene-like fruits, as a ing for and testing morphological synapomorphies for result of abortion of two locules, are a putative these clades will be crucial for their recognition and synapomorphy (Fig. 3) (Hensold, 1991). Nevertheless, understanding. Nevertheless, this search requires the relevance of monospermy as a meaningful char- novel approaches in Eriocaulaceae morphology, which acter would require that the same locule is always is still grounded on Ruhland’s (1903) concepts. fertile in all species belonging to this group, which Certain clades, such as P. subsection Dichocladus, has to be better investigated. represented only by the Brazilian species or the clade As shown in Figure 2, the well-supported mono- comprising Paepalanthus stannardii Giul. & phyletic groups have uniform gynoecium composition, L.R.Parra / Paepalanthus distichophyllus Mart., with either simple fully congenitally fused (1:1) or possess unusual ramification patterns, which could bifid, only basally fused stigmas (1:2). Ruhland (1903) turn out to be synapomorphies. Detailed studies on already noticed the relevance of this character, con- inflorescence morphology could also lead to new per- structing his classification system in a pragmatic way, spectives in the discovery of novel synapomorphies separating the morphologically uniform groups first (Stützel, 1984; Oriani, Scatena & Sano, 2008; Trovó and reordering the variable species under Paepalan- et al., 2010). Two recently reinterpreted, putative thus subgenus Paepalanthus (= P. subgenus Paepalo- morphological synapomorphies for Paepalanthoideae cephalus Ruhland). In this subgenus, Ruhland (1903) were derived from a new developmental framework: found some other uniform groups, such as Paepalan- the central projection of the carpels giving rise to thus section Actinocephalus Koern. (now part of nectariferous branches, and lateral projections of the Actinocephalus), but placed most of the remaining carpels to stigmatic branches. In this context, we species under P. series Paepalanthus (= P. ser. Vari- stress the importance of seeking other unusual and abiles Ruhland). Towards a better understanding of less explored characters, such as embryological and

© 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 171, 225–243 EVOLUTION OF PAEPALANTHOIDEAE 235 developmental characters (Coan & Scatena, 2004; floristic composition is distinct in the two parts of the 2007; Rosa & Scatena, 2007). Espinhaço Range, despite the similar physiognomies and geological origin. Most representatives of Paepalanthoideae inhabit rocky dry soils and are usually confined to a single or BIOGEOGRAPHICAL IMPLICATIONS a few adjacent mountain tops because of limited dis- The occurrence of both subfamilies in Africa and persal ability (Giulietti & Hensold, 1990; Stützel, South America may suggest a Gondwanan origin of 1998). Such constraints might explain the great local Eriocaulaceae, roughly consistent with its crown node diversification of Paepalanthoideae and the elevated estimated at c. 105 Mya (Janssen & Bremer, 2004). number of microendemic species. In clade G, groups Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/1/225/2557485 by guest on 06 January 2020 The initial split of these lineages may have occurred such as Paepalanthus section Diphyomene and shortly before the separation of the continents. Paepalanthus (unranked) Dimeri, have experienced African species of Paepalanthus and Syngonanthus most of their diversification after the dispersal to may be considered relics of the past distribution, cerrado and Lowland Guianan savannas, which is an mainly because the favoured habitats of these genera exception in Eriocaulaceae. Simon et al. (2009) indi- are now rather uncommon in north, north-east and cated, based on molecular dating of various plant south-west Africa. Alternatively, long-distance disper- groups, that diversification in the cerrado may be sal has already been evoked to justify the amphi- recent, not older than 10 Mya, and related to fire Atlantic distribution of many groups; for example, in adaptations. However, without a broader sampling Bromeliaceae (Givnish et al., 2004; Renner, 2004) and and a dated phylogeny it is still difficult to assess Sapotaceae (Bartish et al., 2011). In Eriocaulaceae, whether the habitat restrictions in Eriocaulaceae and long-distance dispersal has also been suggested to the various adaptations to fire are derived from an explain the occurrence of Eriocaulon aquaticum (Hill) ancestor not adapted to fire, as suggested by Simon Druce in North America and in Great Britain (Giuli- et al. (2009). etti & Hensold, 1990). Besides the relatively old age of the Brazilian and In Paepalanthoideae, two main biogeographical the Guiana Shields and the invasion of the savannas, patterns arose from our combined analysis (Fig. 2) a series of events during the Neogene may be respon- and the biogeographical reconstruction (Fig. 5). As in sible for the species diversification in South America Rapateaceae and Bromeliaceae (Givnish et al., 2004), (Antonelli et al., 2009; Hoorn et al., 2010). Hoorn et al. in Eriocaulaceae, we infer that species occurring (2010) correlated Amazonian diversity with the in the Venezuelan–Guayana Highlands are early Andean uplift and its consequences for geology, land- divergent in their lineages. Rondonanthus (all scape evolution and climate change. The diversity of species except R. capillaceus are restricted to the Eriocaulaceae in Amazonia and the Andes is known Venezuelan–Guayana Highlands) is sister to the to be low in comparison with the Espinhaço Range. remaining Paepalanthoideae and P. subgenus Mono- However, such diversity is almost certainly underes- sperma is sister to clade H. The explanation of such a timated, considering both low collecting and taxo- pattern might be found in the advanced age of the nomic efforts (Giulietti & Hensold, 1990). Regarding region and isolation of the populations in the Brazil- the dynamics and the examples described by ian and Guiana Shields. Diversity would be autoch- Antonelli et al. (2009) and Hoorn et al. (2010) for the thonal and derived from isolated ancestries in a region, the inclusion of these species in a dated phy- heterogeneous habitat (Berry & Riina, 2005). Givnish logeny would be relevant in evaluating whether they et al. (2004) pointed to the possibility of mutual colo- are phylogenetically related or not and in reconstruct- nization of both these highlands and the adjacent ing the patterns and routes of colonization of these lowlands, as a result of climate variation. In Erio- and adjacent regions in South and Central America. caulaceae, species diversity is concentrated in the highlands, although there are a few endemic species in the lowlands. Inclusion of these will be essential for FINAL REMARKS AND PERSPECTIVES FOR understanding the processes of local occupation and THE NEXT DECADE diversification (Hensold, 1991, 1999). Although slightly different from previous phyloge- An interesting pattern is revealed in clade H, with netic analyses, our results are strongly supported and the split between a clade formed by plants from the consistent with morphology and with part of the northern region of the Espinhaço Range in Bahia historical classifications. The previous changes in (clade I) and a clade formed by species from the nomenclature proposed for solving issues of Blasto- southern Espinhaço Range in Minas Gerais (clade J) caulon, Syngonanthus and Philodice are further sup- and adjacent mountains. Harley (1988) described a ported (Giulietti et al., 2009; Andrade et al., 2011). similar pattern in Lamiaceae. He suggested that The segregation of Comanthera from Syngonanthus

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evolution of adaptations to fire. Proceedings of the National Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/1/225/2557485 by guest on 06 January 2020 EU924284; Actinocephalus stereophyllus Ruhland Academy of Sciences of the United States of America 106: Sano, MJG Andrade 514 HUEFS, Diamantina/MG, 20359–20364. *1933, EU924437, EU924285; Eriocaulon ligulatum Sokal RR, Rohlf FJ. 1981. Taxonomic congruence in the Lepidomorpha re-examined. Systematic Zoology 30: 309– Vell. L.B. Sm., AM Giulietti 2368 HUEFS, Mucugê/ 325. BA, *298, EU924430, EU924278; Eriocaulon lineari- Staden R, Beal KF, Bonfiel JK. 1998. The Staden package. folium Koern., AM Giulietti 2366 HUEFS, Mucugê/ Methods in Molecular Biology 132: 115–130. BA, *297, EU924429, EU924277; Eriocaulon Stützel T. 1998. Eriocaulaceae. In: Kubitzki K, ed. The fami- modestum Kunth, MJG Andrade 445 HUEFS, Rio de lies and genera of vascular plants, Vol. 4, Monocotyledons: Contas/BA, *780, EU924431, EU924279; Lachnocau- Alismatanae and Commelinanae. Berlin: Springer, 197–207. lon anceps Morong, Goldman s/n BH, Florida/USA, Stützel T. 1984. Blüten und infloreszenzmorphologische *6877, EU924442, –; Leiothrix arrecta Ruhland, AM Untersuchungen zur Systematik der Eriocaulaceen, Vol. 71, Giulietti 2496 HUEFS, Santana do Riacho/MG, Dissertationes Botanicae. Berlin: J. Cramer. *6872, EU924449, EU924296; Leiothrix curvifolia Stützel T. 1985. Die Bedeutung monothecat-bisporangiater Bong. Ruhland, MJG Andrade 553 HUEFS, Datas/ Antheren als systematisches Merkmal zur Gliederung der MG, *1964, EU924446, EU924293; Leiothrix disti- Eriocaulaceen. Botanische Jahrbücher 105: 433–438. choclada Herzog, MJG Andrade 458 HUEFS, Rio de Swofford DW. 2002. PAUP*: phylogenetic analysis using Contas/BA, *792, EU924447, EU924294; Leiothrix parsimony * and other methods, ver. 4 beta 10. Sunderland, flagellaris Guill. Ruhland, MJG Andrade 485 MA: Sinauer Associates. HUEFS, Grão-Mogol/MG, *1920, EU924450, Taberlet P, Gielly L, Pautou G, Bouvet J. 1991. Universal EU924297; Leiothrix flavescens Bong. Ruhland, MJG primers for amplification of three non-coding regions of Andrade 440 HUEFS, Rio de Contas/BA, *775, chloroplast DNA. Plant Molecular Biology 17: 1105–1109. EU924444, EU924291; Leiothrix vivipara Bong. Tissot-Squalli HML. 1997. Monographische Bearbeitung Ruhland, AM Giulietti 2503 HUEFS, Santana do von Paepalanthus subgenus Platycaulon, Vol. 280, Disserta- Riacho/MG, *7415, EU924451, EU924298; Paepalan- tiones Botanicae. Berlin: J. Cramer. thus acantholimon Ruhland, MLO Trovó 250 SPF, Trovó M, Stützel T, Scatena VL, Sano PT. 2010. Morphol- ogy and anatomy of inflorescence and inflorescence axis in Alto Caparaó/MG, *6924, GQ475202, GQ475232; Paepalanthus sect. Diphyomene Ruhland (Eriocaulaceae), Paepalanthus acanthophyllus Ruhland, MLO Trovó Poales and its taxonomic implications. Flora 205: 242–250. 287 SPF, Alto Paraíso de Goiás/GO, *6908, Unwin MM. 2004. Molecular systematics of Eriocaulaceae GQ475203, GQ475233; Paepalanthus acuminatus Martynov. Unpublished D. Phil. Thesis, Miami University. Ruhland, MLO Trovó 175 SPF, Lima Duarte/MG, Varanda EA, Varela SD, Rampazo RA, Kitagawa RR, *6928, GQ475204, GQ475234; Paepalanthus albidus Raddi MSG, Vilegas W, Santos LC. 2006. Mutagenic and Gardner, MJG Andrade 541 HUEFS, Diamantina/ cytotoxic effect of planifolin: a naphthopyranone dimer iso- MG, *1953, EU924439, EU924287; Paepalanthus are- lated from Paepalanthus planifolius. Toxicology in Vitro 20: tioides Ruhland, MLO Trovó 357 SPF, Diamantina/ 664–668. MG, *6914, GQ475205, GQ475235; Paepalanthus Yu Y, Harris AJ, He XJ. 2011. RASP: reconstruct ancestral argenteus Bong. Koern., MJG Andrade 539 HUEFS, state in phylogenies, ver. 1.1. Available at: http://mnh.scu. Diamantina/MG, *1952, EU924484, EU924331; edu.cn/soft/blog/RASP Paepalanthus augustus Silveira, LM Borges 178 SPF, Santana do Riacho/MG, *6963, –, GQ475236; Paepal- anthus bifidus Scharad. Kunth, MJG Andrade 489 HUEFS, Grão-Mogol/MG, *1924, EU924453, APPENDIX 1 EU924300; Paepalanthus bonsai Trovó and Sano, PL Voucher information and GenBank accession Vianna 2776 SPF, São Gonçalo do Rio Preto/MG, numbers for taxa used in this study. An asterisk *6903, GQ475206, GQ475237; Paepalanthus bro- indicates the number in the HUEFS genetic bank. melioides Silveira, MLO Trovó 219 SPF, Santana do A dash indicates the molecular region was not Riacho/MG, *6905, GQ475207, GQ475238; Paepalan- sampled. thus bryoides Kunth, FN Costa 263 HUEFS,

Diamantina/MG, *6878, EU924452, EU924299; GQ475220, GQ475251; Paepalanthus microphyllus Paepalanthus calvoides Ruhland, MJG Andrade Kunth, MLO Trovó 225 SPF, Santana do Riacho/MG, 550 HUEFS, Itacambira/MG, *6812, GQ475208, *6923, GQ47522, GQ475252; Paepalanthus mollis GQ475239; Paepalanthus canescens Koern., MLO Kunth, MLO Trovó 376 SPF, Diamantina/MG, *6961, Trovó 351 SPF, Diamantina/MG, *6919, GQ475209, GQ475222, GQ475253; Paepalanthus myocephalus GQ475240; Paepalanthus caparoensis Ruhland, MLO Mart., MJG Andrade 613 HUEFS, Feira de Santana/ Trovó 249 SPF, Alto Caparaó/MG, *6925, GQ475210, BA, *6879, EU924454, EU924301; Paepalanthus GQ475241; Paepalanthus chrysophorus Silveira, MM neglectus Koern., BRN Araújo 85 HUEFS, Rio de Lopes 951 SPF, Grão-Mogol/MG, *6958, GQ475211, Contas/BA, *1805, EU924460, EU924308; Paepalan- GQ475242; Paepalanthus cinereus Giulietti and thus nigrescens Silveira, MLO Trovó 204 SPF, Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/1/225/2557485 by guest on 06 January 2020 Parra, AM Giulietti s/n HUEFS, Rio de Contas/BA, Santana do Riacho/MG, *6932, GQ475223, *1293, EU924469, EU924316; Paepalanthus comans GQ475254; Paepalanthus obtusifolius Koern., R Silveira, MJG Andrade 540 HUEFS, Diamantina/ Harley 54802 HUEFS, Rio de Contas/BA, *1321, MG, *6820, EU924482, EU924329; Paepalanthus cor- EU924457, EU924304; Paepalanthus parviﬂorus datus Ruhland, MLO Trovó 443 SPF, Alto Paraíso de Hensold Hensold, MLO Trovó 202 SPF, Santana do Goiás/GO, *6917, GQ475212, GQ475243; Paepalan- Riacho/MG, *6962, GQ475224, –; Paepalanthus thus cylindraceus Silveira, MLO Trovó 383 SPF, São pedunculatus Ruhland, MJG Andrade 547 HUEFS, Roque de Minas/MG, *9262, –, GQ475244; Paepalan- Datas/MG, *1958, GQ475225, GQ475255; Paepalan- thus distichophyllus Mart., MLO Trovó 218 SPF, thus planifolius Bong. Koern., MJG Andrade 526 Diamantina/MG, *6907, GQ475213, GQ475245; HUEFS, Diamantina/MG, *1942, EU924485, Paepalanthus elongatus Bong. Koern., MJG Andrade EU924332; Paepalanthus polycladus Silveira, MLO 572 HUEFS, Tiradentes/MG, *6884, EU924467, Trovó 391 SPF, São Roque de Minas/MG, *6909, EU924314; Paepalanthus erigeron Mart. ex Koern., GQ475226, GQ475256; Paepalanthus polygonus AA Ribeiro-Filho 107 HUEFS, Lençóis/BA, *6882, –, Koern., MLO Trovó 413 SPF, Diamantina/MG, *6913, EU924306; Paepalanthus eriophaeus Ruhland, MJG GQ475227, GQ475257; Paepalanthus pulchellus Andrade 504 HUEFS, Itacambira/MG, *6811, Herzog, AM Giulietti 2423 HUEFS, Rio de Contas/BA, EU924459, EU924307; Paepalanthus exiguus Bong. *1409, EU924461, EU924309; Paepalanthus pulvina- Koern., E Guarçoni 710 HUEFS, Alto Caparaó/MG, tus N.E. Br., R Harley 54634 HUEFS, Mucugê/BA, *6885, EU924481, EU924328; Paepalanthus frater- *638, EU924465, EU924313; Paepalanthus regalis nus N.E. Br., P Fiaschi 3202 SPF, Mount Roraima/ Mart., R Harley 54640 HUEFS, Mucugê/BA, *660, Venezuela, *6912, GQ475214, GQ475246; EU924462, EU924310; Paepalanthus repens Koern., Paepalanthus fulgidus Moldenke, P Fiaschi 3196 R. Abbott 21006 FLAS, Dominican Republic, *7416, SPF, Mount Roraima/Venezuela, *6911, GQ475215, EU924474, EU924321; Paepalanthus rupestris GQ475247; Paepalanthus geniculatus Bong. Kunth, Gardner, MJG Andrade 542 HUEFS, Diamantina/ MLO Trovó 205 SPF, Diamantina/MG, *6921, MG, *1954, EU924440, EU924288; Paepalanthus GQ475216, GQ475248; Paepalanthus giganteus Sano, scirpeus Mart. ex Koern. Giulietti, JR Pirani 4162 MJG Andrade 527 HUEFS, Diamantina/MG, *1943, HUEFS, Congonhas do Norte/MG, *6876, EU924441, EU924478, EU924325; Paepalanthus glareosus EU924289; Paepalanthus scleranthus Ruhland, MJG Kunth, MJG Andrade 548 HUEFS, Datas/MG, *1959, Andrade 537 HUEFS, Diamantina/MG, *1951, EU924475, EU924322; Paepalanthus glaziovii EU924410, EU924335; Paepalanthus sessiliﬂorus Ruhland, Sano 3851 SPF, Diamantina/MG, *6902, Mart. ex Koern., JG Jardim 2237 HUEFS, Maraú/ GQ475217, GQ475249; Paepalanthus henriquei BA, *6881, EU924458, EU924305; Paepalanthus sil- Ruhland, MLO Trovó 179 SPF, Lima Duarte/MG, veirae Ruhland, MJG Andrade 568 HUEFS, *6927, GQ475218, –; Paepalanthus implicatus Sil- Tiradentes/MG, *1976, EU924463, EU924311; veira, MJG Andrade 550 HUEFS, Datas/MG, *1961, Paepalanthus spathulatus Koern., R Harley 55476 EU924472, EU924319; Paepalanthus klotzschianus HUEFS, Itaberaba/BA, *6883, EU924464, EU924312; Koern., MLO Trovó 257 SPF, Linhares/ES, *6959, Paepalanthus sphaerocephalus Ruhland, MJG GQ475219, GQ475250; Paepalanthus lamarckii Andrade 456 HUEFS, Rio de Contas/BA, *790, Kunth, DS Carneio-Torres 461 HUEFS, Lagartos/SE, EU924480, EU924327; Paepalanthus stannardii *6880, EU924456, EU924303; Paepalanthus leuco- Giulietti and Parra, MJG Andrade 438 HUEFS, Rio cephalus Ruhland, MJG Andrade 620 HUEFS, Rio de de Contas/BA, *773, EU924473, EU924320; Paepal- Contas/BA, *6886, EU924487, EU924334; Paepalan- anthus strictus Koern., MJG Andrade 491 HUEFS, thus macrocaulon Silveira, MJG Andrade 431 Grão-Mogol/MG, *1926, EU924471, EU924318; HUEFS, Rio de Contas/BA, *766, EU924470, Paepalanthus subcaulescens N.E. Br., P Fiaschi 3195 EU924317; Paepalanthus macropodus Ruhland, MLO SPF, Mount Roraima/Venezuela, *6957, GQ475228, Trovó 214 SPF, Santana do Riacho/MG, *6922, GQ475258; Paepalanthus superbus Ruhland, AM

Giulietti 2504 HUEFS, Santana do Riacho/MG, donanthus capillaceus Koern. Hensold and Giulietti, *6858, EU924483, EU924330; Paepalanthus tortilis van den Berg 1792 HUEFS, Mount Roraima/ Bong. Mart., MJG Andrade 479 HUEFS, Grão-Mogol/ Venezuela, *6889, GQ478282, EU924338; Rondonan- MG, *1914, EU924455, EU924376; Paepalanthus tri- thus roraimae Oliver Herzog, P Fiaschi 3201 SPF, chophyllus Bong. Koern., MJG Andrade 439 HUEFS, Mount Roraima/Venezuela, *6916, GQ475231, –; Rio de Contas/BA, *774, EU924479, EU924326; Syngonanthus aciphyllus Bong. Ruhland, MJG Paepalanthus tuberosus Bong. Kunth, van den Berg Andrade 532 HUEFS, Diamantina/MG, *1948, 1364 HUEFS, Conceição do Mato Dentro/MG, *1641, EU924491, EU924339; Syngonanthus arenarius EU924486, EU924333; Paepalanthus urbanianus Gardner Ruhland, MJG Andrade 493 HUEFS, Ruhland, MLO Trovó 435 SPF, Alto Paraíso de Goiás/ Itacambira/MG, *6802, EU924498, EU924342; Syngo- Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/1/225/2557485 by guest on 06 January 2020 GO, *6918, –, GQ475259; Paepalanthus vaginatus nanthus caulescens Poir. Ruhland, MJG Andrade 616 Koern., MLO Trovó 353 SPF, Diamantina/MG, *6904, HUEFS, Rio de Contas/BA, *786, EU924500, –, GQ475260; Paepalanthus vellozioides Koern., MLO EU924344; Syngonanthus curralensis Moldenke, Trovó 198 SPF, Santana do Riacho/MG, *6906, MJG Andrade 595 HUEFS, Rio de Contas/BA, *6890, GQ475229, GQ475261; Paepalanthus viridulus EU924492, EU924340; Syngonanthus vernonioides Ruhland, C Sarquis 12 SPF, Lima Duarte/MG, *6915, Kunth Ruhland, AM Giulietti 2185 HUEFS, Rio de GQ475230, GQ475262; Philodice hoffmannseggii Contas/BA, *220, EU924499, EU924343; Tonina ﬂu- Mart., AM Giulietti 2483 HUEFS, Nossa Senhora do viatilis Aubl., MJG Andrade 616 HUEFS, Recife/PE, Livramento/MT, *6887, EU924411, EU924336; Ron- *6895, EU924501, EU924345.

APPENDIX 2 MORPHOLOGICAL CHARACTERS WITH THEIR RESPECTIVE STATES AND THE MORPHOLOGICAL MATRIX (1) Stigmatic branches: 0 carinal single, 1 commissural single, 2 commissural bifid; (2) glands on staminate flower: 0 present, 1 absent; (3) androecium: 0 diplostemonous, 1 haplostemonous; (4) leaf fenestration: 0 present 1 absent; (5) petals of the staminate flower: 0 free, 1 tubular; (6) linear staminodes on pistillate flower: 0 absent, 1 present; (7) fertile locules of the gynoecium: 0 all, 1 only the median; (8) petals of the pistillate flower: 0 free, 1 fused at the middle; (9) trichomes inside the corolla of the staminate flower: 0 absent, 1 present; (10) involucral bracts adaxial surface: 0 glabrous, 1 hairy; (11) scapes: 0 free, 1 fused; (12) involucral bracts: 0 not surpassing the capitula, 1 surpassing the capitula; (13) floral merism: 0 trimerous, 1 dimerous.

Taxa/character 12345678910111213

Eriocaulon linearifolium 0000000000 0 0 0 Eriocaulon ligulatum 0000000000 0 0 0 Eriocaulon modestum 0000000000 0 0 0 Rondonanthus capillaceus 2111010000 0 0 0 Leiothrix distichoclada 1111100000 0 0 0 Leiothrix flavescens 1111100000 0 0 0 Leiothrix curvifolia 1111100000 0 0 0 Leiothrix flagellaris 1111100000 0 0 0 Leiothrix arrecta 1111100000 0 0 0 Leiothrix vivipara 1111100000 0 0 0 Philodice hoffmannseggii 1111100100 0 0 0 Syngonanthus arenarius 1111100100 0 1 0 Syngonanthus caulescens 1111100100 0 0 0 Comanthera vernonioides 1111100100 0 0 0 Comanthera aciphylla 1111100100 0 1 0 Comanthera curralensis 1111100100 0 1 0 Tonina fluviatilis 2111100000 0 0 0 Paepalanthus lamarckii 2111100000 0 0 0 Paepalanthus obtusifolius 2111100000 0 0 0 Paepalanthus tortilis 2111100000 0 0 0 Paepalanthus repens 2111100000 0 0 0 Paepalanthus distichophyllus 1111100000 0 0 0 Paepalanthus stannardii 1111100000 0 0 0 Paepalanthus exiguus 1111100000 0 0 1

APPENDIX 2 Continued

Taxa/character 12345678910111213

Paepalanthus 1111100000 0 0 1 leucocephalus Paepalanthus scleranthus 1111100000 0 0 1 Actinocephalus bongardii 1111100000 0 0 0 Actinocephalus ramosus 1111100000 0 0 0 Paepalanthus geniculatus 1111100000 0 0 0 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/1/225/2557485 by guest on 06 January 2020 Paepalanthus glareosus 1111100000 0 0 0 Actinocephalus stereophyllus 1111100000 0 0 0 Actinocephalus brachypus 1111100000 0 0 0 Actinocephalus ciliatus 1111100000 0 0 0 Paepalanthus elongatus 2111100000 0 0 1 Paepalanthus strictus 2111100000 0 0 1 Paepalanthus trichophyllus 2111100000 0 0 1 Paepalanthus polycladus 2111100000 0 0 1 Paepalanthus giganteus 2111100000 0 0 1 Paepalanthus 2111100000 0 0 1 acanthophyllus Paepalanthus cordatus 2111100000 0 0 1 Paepalanthus subcaulescens 2111101000 0 0 0 Paepalanthus fraternus 2111101000 0 0 0 Paepalanthus fulgidus 2111101000 0 0 0 Paepalanthus cinereus 2111100000 0 0 0 Paepalanthus macrocaulon 2111100000 0 0 0 Paepalanthus neglectus 2111100000 0 0 0 Paepalanthus regalis 2111100000 0 0 0 Paepalanthus spathulatus 2111100000 0 0 0 Paepalanthus klotzschianus 2111100000 0 0 0 Paepalanthus pulchellus 2111100000 0 0 0 Paepalanthus pulvinatus 2111100000 0 0 0 Paepalanthus albidus 2111100000 0 0 0 Paepalanthus rupestris 2111100000 0 0 0 Paepalanthus bonsai 2111100000 0 0 0 Paepalanthus glaziovii 2111100000 0 0 0 Paepalanthus nigrescens 2111100010 0 0 0 Paepalanthus argenteus 2111100011 0 1 0 Paepalanthus comans 2111100011 0 1 0 Paepalanthus superbus 2111100011 0 1 0 Paepalanthus mollis 2111100011 0 1 0 Paepalanthus macropodus 2111100000 1 0 0 Paepalanthus bromelioides 2111100000 1 0 0 Paepalanthus planifolius 2111100000 1 0 0 Paepalanthus vellozioides 2111100000 1 0 0 Paepalanthus tuberosus 2111100000 1 0 0 Paepalanthus polygonus 2111100000 0 0 0 Paepalanthus 2111100000 0 0 1 sphaerocephalus Paepalanthus viridulus 2111100000 0 0 0 Paepalanthus chrysophorus 2111100000 0 0 0 Paepalanthus calvoides 2111100000 0 0 0 Paepalanthus eriophaeus 2111100000 0 0 0 Paepalanthus acantholimon 2111100000 0 0 0 Paepalanthus caparoensis 2111100000 0 0 0 Paepalanthus acuminatus 2111100000 0 1 0 Paepalanthus silveirae 2111100000 0 0 0 Paepalanthus scirpeus 1111100000 0 0 0 Paepalanthus bryoides 1111100000 0 0 0 Paepalanthus aretioides 2111100000 0 0 0 Paepalanthus microphyllus 2111100000 0 0 0

APPENDIX 2 Continued

Taxa/character 12345678910111213

Paepalanthus implicatus 2111100000 0 0 0 Paepalanthus canescens 2111100000 0 0 0 Paepalanthus pedunculatus 2111100000 0 0 0

APPENDIX 3 Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/1/225/2557485 by guest on 06 January 2020 Geographical distribution matrix according to the following areas: (A) Espinhaço Range in Minas Gerais; (B) Espinhaço Range in Bahia; (C) cerrado; (D) Lowland Guianan savannas; (E) restingas; (F) tepuis; and (G) Caribbean. 0 = absent; 1 = present.

Taxa/area A B C D E F G

Eriocaulon linearifolium 0110000 Eriocaulon ligulatum 1110000 Eriocaulon modestum 1110000 Rondonanthus capillaceus 0001000 Leiothrix distichoclada 0100000 Leiothrix flavescens 1111110 Leiothrix curvifolia 1000000 Leiothrix flagellaris 1000000 Leiothrix arrecta 1000000 Leiothrix vivipara 1000000 Philodice hoffmannseggii 0010000 Syngonanthus arenarius 1000000 Syngonanthus caulescens 1111111 Comanthera vernonioides 1100000 Comanthera aciphylla 1000000 Comanthera curralensis 0100000 Tonina fluviatilis 0000100 Paepalanthus lamarckii 0000100 Paepalanthus obtusifolius 0100000 Paepalanthus tortilis 1111111 Paepalanthus repens 0000001 Paepalanthus distichophyllus 1000000 Paepalanthus stannardii 0100000 Paepalanthus exiguus 1000000 Paepalanthus leucocephalus 1000000 Paepalanthus scleranthus 1000000 Actincephalus bongardii 1110000 Actinocephalus ramosus 1100100 Paepalanthus geniculatus 1000000 Paepalanthus glareosus 1000000 Actinocephaus stereophyllus 1000000 Actinocephalus brachypus 1000000 Actinocephalus ciliatus 1000000 Paepalanthus elongatus 1110000 Paepalanthus strictus 1100000 Paepalanthus trichophyllus 1110000 Paepalanthus polycladus 1000000 Paepalanthus giganteus 1011000 Paepalanthus acanthophyllus 0010000 Paepalanthus cordatus 0010000 Paepalanthus subcaulescens 0000010 Paepalanthus fraternus 0000010

APPENDIX 3 Continued

Taxa/area A B C D E F G

Paepalanthus fulgidus 0000010 Paepalanthus cinereus 0100000 Paepalanthus macrocaulon 0100000 Paepalanthus neglectus 0100000 Paepalanthus regalis 1100000

Paepalanthus spathulatus 0100000Downloaded from https://academic.oup.com/botlinnean/article-abstract/171/1/225/2557485 by guest on 06 January 2020 Paepalanthus klotzschianus 0000100 Paepalanthus pulchellus 0100100 Paepalanthus pulvinatus 0100000 Paepalanthus albidus 1000000 Paepalanthus rupestris 1000000 Paepalanthus bonsai 1000000 Paepalanthus glaziovii 1000000 Paepalanthus nigrescens 1000000 Paepalanthus argenteus 1000000 Paepalanthus comans 1000000 Paepalanthus superbus 1000000 Paepalanthus mollis 1000000 Paepalanthus macropodus 1000000 Paepalanthus bromelioides 1000000 Paepalanthus planifolius 1000000 Paepalanthus vellozioides 1000000 Paepalanthus tuberosus 1000000 Paepalanthus polygonus 1000000 Paepalanthus sphaerocephalus 1110000 Paepalanthus viridulus 1000000 Paepalanthus chrysophorus 1000000 Paepalanthus calvoides 1000000 Paepalanthus eriophaeus 1000000 Paepalanthus acantholimon 1000000 Paepalanthus caparoensis 1000000 Paepalanthus acuminatus 1000000 Paepalanthus silveirae 1000000 Paepalanthus scirpeus 1000000 Paepalanthus bryoides 1000000 Paepalanthus aretioides 1000000 Paepalanthus microphyllus 1000000 Paepalanthus implicatus 1000000 Paepalanthus canescens 1000000 Paepalanthus pedunculatus 1000000

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Appendix S1. The 50% majority rule consensus tree of the Bayesian analysis of the trnL-trnF data set. Appendix S2. The 50% majority rule consensus tree of the Bayesian analysis of the ITS data set.